Plant Disease

Insecticide applications are commonly recommended for managing Diaphorina citri, the vector of huanglongbing (HLB), but their effectiveness in reducing transmission of ‘Candidatus Liberibacter asiaticus’ (CLas), especially during continuous psyllid influx and shoot growth, remains unclear. This study evaluated the efficacy of foliar application of thiamethoxam and spinetoram in reducing CLas transmission in sweet orange seedlings. Two experiments were conducted up to 13th days after first spray. In experiment #1, CLas-positive psyllids were confined in seedlings every two days. Treatments were: control; S14 — one spray at day 0; and S7 — sprays at days 0 and 7. In experiment #2, psyllids were released for free choice within a screenhouse every two days. Treatments were: control; S14; S7; and S3 — sprays at day 0 and every 3 days. Sprays on day 0 were over unfolded leaves (V2 stage). Psyllid mortality was assessed in experiment #1. Psyllid occupancy (percentage of seedlings with at least one psyllid) and abundance (number of psyllids per seedling) were assessed in experiment #2. After 6 months, seedlings were tested for CLas by qPCR. In experiment #1, cumulative psyllid mortality was higher in sprayed treatments than control for both insecticides, but HLB incidence was similar. In experiment #2, both psyllid occupancy and abundance decreased with shorter spray intervals, with the lowest HLB incidence in S3. These findings suggest that reducing spray intervals can limit CLas transmission during periods of shoot growth and continuous vector pressure. However, CLas transmission was not entirely prevented, highlighting the need for integrated, area-wide management strategies.

‘Candidatus Liberibacter asiaticus’ (CLas), the agent associated with the Huanglongbing citrus disease, is commonly detected using quantitative PCR (qPCR) with hydrolysis probes. Internal standards are typically included in the qPCR assays to reduce the risk of false negatives caused by inhibitors. When the internal standard is detected but CLas is not, it is generally assumed that the pathogen is absent from the tested sample. However, our study shows that trace amounts of CLas may go undetected if the internal standard is either overly abundant or too dissimilar to CLas. To overcome these limitations, we developed a synthetic internal standard (IS) that uses the same primer as the CLas target sequence, along with three to four downstream nucleotides, but with a unique internal sequence derived from smooth hammerhead shark (Sphyrna zygaena; IS-SHK). This sequence matches the guanine-cytosine content and melting temperature of the CLas target and was specifically selected to minimize potential interference from other nucleic acid materials in citrus samples. To minimize competition between the IS-SHK standard and the CLas target, an average of only 21 molecules of IS-SHK standard is added to each qPCR reaction. Therefore, when IS-SHK standard is detected at expected levels and CLas is not, the absence of CLas is confirmed rather than inhibition of the detection. Conversely, the absence of both IS-SHK standard and CLas suggests the presence of a qPCR inhibitor, warranting retesting of the sample.